Recombination and Transposition
(Watson, Baker, Bell, Gann, Levine and Losick bab 11, Yuwono bab
13, Weaver Bab 22 &23)
Dr. Ratna Megawati Widharna, SKG, MFT
Mutation, Recombination
Mutation: DNA structural/ composition change in genome of individual that can cause phenotipic change in the individu, although not every mutation can change the phenotypeGenetic recombination : genetic element exchange process that occur between different DNA strand (interstrand) or between gen parts located in 1 DNA strand (intrastrand)Recombination
Started by both homolog chromosom DNA strand cutting on the same site by nuclease activity
In Escherichia coli, nuclease is coded by gen recB and recC which are responsible in exonuclease V (135 kDalton) and exonuclease V (125 kDalton)
Cutting only occur in one of both chromosom DNA double strand
3 genetic recombinant
General/Homolog recombinantSite-specific recombinationTransposition/replicative recombinationRecombinant DNA construction
Trudy McKee, Biochemistry : The molecular Basis of LifeHomolog recombination
Cause the exchange DNA intermolecule that has quite big homology of nucleotide sequence Specific character : the process can occur in every point in homology areaRecombination occur through the DNA strand direct breakage which is followed by the rejoining processHomolog recombination
Interchromosom recombination involves physical exchange process between chromosom parts
Recombination process takes place accurately no single nucleotide base pair is missing or included in recombinant chromosom
Structure seen as chiasma in meiosis
Chiasma = cutting and splicing DNA strand, when 2 non-sister chromatids is cut and joined one another
Key steps of homolog recombination
Allignment of 2 homologous DNA molecules (DNA sequences are identical / nearly identical for a region of at least 100 base pairs / so). Despite this high degree of similarity, DNA molecules can have small regions of sequence difference and may carry different sequence variants, known as alleles, of the same gene
Key steps of Homologous recombination (2)
2. Introduction of breaks in the DNABreaks may occur in 1 DNA strand / involve both DNA standsThe nature of these breaks = feature that largely distinguishes the 2 models (site specific and transposition recombination)Key steps of Homologous recombination (3)
3. Formation of initial short regions of base pairing between the 2 recombining DNA molecules. This pairing occurs when a single-stranded region of DNA originating from one parental molecule pairs with its complementary strand in the homologous duplex DNA molecule Strand invasion the 2 DNA molecules become connected by crossing DNA strands Holliday junction
Key steps of Homologous recombination (4)
4. Movement of the Holliday junction
A Holliday junction can move along the DNA by the repeated melting and formation of base pairs
Each time the junction moves, base pairs are broken in the parental DNA molecules while identical base pairs are formed in the recombination intermediate Branch migration
Key steps of Homologous recombination (5)
5. Cleavage of the Holliday junction
Cutting the DNA strands within the Holliday junction regenerates 2 separate duplex DNA molecules finishes genetic exchange RESOLUTION
Which of the 2 pairs of DNA strands in the Holliday juction are cut during resolution has a large impact on the extent of DNA exchange that occurs between the 2 recombining molecules
Homolog recombination
Started when 2 homolog chromosom is located near each other so that the homolog nucleotide sequence can be exchanged.Synapsis = contact between the 2 chromosom pairs in ProphaseModel HollidayInvasion is symmetrical: the same region of DNA sequence is swapped between the 2 molecules
Strand invasion generates the Holliday junction, the key recombination intermediate
Holliday junction generated by strand invasion can then move along the DNA by branch migration increases the length of DNA exchanged
Heteroduplex DNA
If the 2 DNA molecules are not identical-but, e.g., carry a few small sequence differences, as is true often between 2 alleles of the same gene-branch migration through these regions of sequence difference generates DNA duplexes carrying 1 or a few sequence mismatches (B and b alleles in Fig 10-1d & inset) HETERODUPLEX DNAFinishing recombination (resolution of the Holliday junction)
Fig 10-2: alternative pairs of DNA cut sites occur on the branched DNATo make these cut sites easier to visualize, the Holliday junction is rotated to give a square-planner structure with no crossing strandsThe 2 alternative choices for cleavage sites : 1 and 2 (Fig 10-2)Splice recombination
The cut sites marked 1 occur in the 2 DNA strands that were not broken during the initiation reaction (Fig 10-1b)
If these strands are now cut, ant then covalently joined (the 2nd reaction catalyzed by DNA ligase), the resulting DNA molecules will have the structure and sequence shown in the left bottom of the figure
Splice recombination products because the 2 original duplexes are now spliced together such that regions from the parental DNA molecules are covalently joined together by a region of hybrid duplex
Crossover product
Generation of splice products results in reassortment of genes that flank the site of recombination crossover product within this DNA molecule, crossing over has occurred between the A and C genesPatch products
In contrast, the alternative pair of cut sites in the Holliday junction (marked 2 in Fig 10-2) is in the 2 DNA strands that were broken to initiate recombination. After resolution and covalent joining of the strands at these sites, the resulting DNA molecules contain a region or patch of hybrid DNA Patch products
Recombination does not result in reassortment of the genes flanking the site of initial cleavage (see fate of the A/a and C/c allele markers) non-crossover products
Holliday junction cleavage
Holliday junction cleavage
Double-Strand Break Repair Model More Accurately Describes Many Recombination Events
Homologous recombination is often initiated by double-stranded breaks in DNA double-stranded break repair pathway (Fig 10-3)
Holliday model : starts with aligned homologous chromosomes
Initiating event : introduction of a double-stranded break (DSB) in 1 of the 2 DNA molecules (Fig 10-3a). The other DNA duplex remains intact
After introduction of the DSB, a DNA-cleaving enzyme sequentially degrade the broken DNA molecule to generate the regions of single-strand stranded DNA (Fig 10-3b) creates single-strand extensions (ssDNA tails) on the broken molecules terminate with 3 ends
In some cases, both strands at a DSB r processed, whereas in other cases, only the 5-terminating strand is degraded
The ssDNA tails generated then invade the unbroken homologous DNA duplex (Fig 10-3c)
Fig: 1 strand invasion, as likely occurs initially
Next panel : 2 invading strands
In each case, the invading strand base-pairs with its complementary strand in the other DNA molecules
Because the invading strands ends with 3 termini, they can serve as primers for new DNA synthesis
gene conversion event
If the 2 original DNA duplexes were not identical in sequence near the site of the break (e.g, having single base-pair changes), sequence information could be lost during recombination by the DSB-repair pathway.
Fig10-3: sequence information lost from the gray DNA molecule as a result of DNA processing is replaced by the sequence present on the blue duplex as a result of DNA synthesis
These nonreciprocal step in DSB-repair sometimes leaves a genetic trace-giving rise to a gene conversion event
The 2 Holliday junctions found in the recombination intermediates generated by this model move by branch migration and ultimately are resolved to finish recombination
Once again, the strands that are cleaved during resolution of these Holliday junctions determine whether the product DNA molecules will contain reassorted genes in the regions flanking the site of recombination (i.e result in crossing over) or not
For each junction, there are 2 possible cleavage sites (site 1 and site 2)
If both junctions are cleaved in the same way, that is either both at site 1 or both at site 2, then non-crossover products will be generated (panel b of the figure).
Notice, the allele markers A/B and /b are still on the same DNA molecules as they were in the parental chromosomes.
Cleavage of both junctions at site 1 : non-crossover products
In contrast, when 2 Holliday junction are cleaved using different sites, crossover products are generated (panel C of figure)
Junction x was cleaved at site 1 whereas junction y was cleaved at site 2
Notice that now gene A is linked to gene b, whereas gene a is linked to gene B; thus reassortment of the flanking gene has occurred.
Cleavage of junction x at site 2 and junction y at site 1 also generates crossover products
Simple rule (1)
Cleavage at both junctions at site 2 will give a patch product (patch + patch, non-crossover products)Cleavage at both junctions at site 1 also gives a patch product (splice + splice = patch because the 2nd splice-type resolution essentially underdoes the rearrangement caused by the 1st cleavageSimple rule (2)
Cleavage of 1 junction at site 1, but the other at site 2 therefore generates crossover products (splice + patch = splice) because the arrangement caused by the site 1 cleavage is retained in the final productFunction of homologous recombination in bacteria
To repair double-stranded breaks in DNA ( ~ eucaryotic cell)To restart collapsed replication forks ( ~ eucaryotic cell)To allow a cells chromosomal DNA to recombine with DNA that enters via phage infection/conjugationHomologous recombination in Eucaryotes
Cells with defects in the proteins that promote recombination are hypersensitive to DNA damaging agents, esp those that break DNA strands
Animal carrying mutations that interfere with homologous recombination are predisposed to certain types of cancer
Critical for meiosis required for proper chromosome pairing maintaining integrity of the genome
Recombination reshuffles genes between the parental chromosomes, ensuring variation in the sets of genes passed to the next generation
Homologous Recombination is Required for Chromosome Segregation during Meiosis
Before division: cell has 2 copies of each chromosome (the homologs) 1 each that was ingerited from its 2 parents
During the S phase, these chromosomes are replicated to give a total DNA content of 4N
The products of replication i.e. sister chromatids-stay together
Then, in the preparation for the 1st nuclear division, these duplicated homologous chromosomes must pair & align & then separate sister chromatin remain paired
Then, in the 2nd nuclear division, it is the sister chromatids that separate product : 4 gametes, each with 1 copy of 2 chromosome (i.e. the 1N DNA content)
Without recombination, chromosomes often fail to align properly for the first meiotic division a high incidence of chromosome loss improper segregation of chromosomes: NONDISJUNCTION
Nondisjunction
Leads to a large number of gametes without the correct chromosome complement
Gametes with either too few / too many chromosomes cannot develop properly once fertilized failure in homologous recombination poor fertility
Homologous recombination events that occur during meiosis : Meiotic recombination frequently give rise to crossing over between genes on the 2 homologous parental chromosomes
Site-Specific Recombination & Transposition of DNA
Dr. Ratna Megawati Widharna, SKG, MFT
Molecular biology
References
Watson, Baker, Bell, Gann, Levine and Losick bab 11, Yuwono bab 13, Weaver Bab 22 &23
Although DNA replication, repair, homologous recombination occur with high fidelity to ensure the genome identity between generations, there are genetic processes that rearrange DNA sequences and thus lead to a more dynamic genome structure
Two classes of genetic recombination for DNA rearrangement:
Conservative site-specific recombination (CSSR): recombination between two defined sequence elementsTranspositional recombination (Transposition): recombination between specific sequences and nonspecific DNA sitesFigure 11-1
OUTLINE
Conservative Site-Specific Recombination
Biological Roles of Site-Specific Recombination (l phage integration/excision, multimeric genome resolution)
Transposition ( concepts, learning from B. McClintock, DNA tranposons. Viral-like retrotransposons/retroviruses, poly-A retrotransposons)
Topic 1: Conservative Site-Specific Recombination
Exchange of non-homologous sequences at specific DNA sites(what) Mediated by proteins that recognize specific DNA sequences. (how)Conservative Site-Specific Recombination
1-1 Site-specific recombination occurs at specific DNA sequences in the target DNA
CSSR can generate three different types of DNA rearrangements
Figure 11-3
If the two sites at which recombination will take place are oriented oppositely to one another in the same DNA molecule then the site-specific reacombination results in inversion of the segment of DNA between the two recombination sites
recombination at inverted repeats causes an inversion
If the two sites at which recombination will take place are oriented in the same direction in the same DNA molecule, then the segment of DNA between the two recombinogenic sites is deleted from the rest of the DNA molecule and appears as a circular molecule. Insertion is the reverse reaction of the deletion
recombination at direct repeats causes a deletion
Figure 11-4 Structures involved in CSSR
Conservative Site-Specific Recombination
1-2 Site-specific recombinases cleave and rejoin (join) DNA using a covalent protein-DNA intermediate
Figure 11-5
The covalent protein-DNA intermediate conserves the energy of the cleaved phosphodiester bond within the protein-DNA linkage, which allows the cleaved DNA strands to be rejoined/resealed by reversal of the the cleavage process
This mechanistic feature contributes the conservative to the CSSR name, because every DNA bond that is broken during the reaction is resealed by the recombinase without consuming any external energy.
Conservative Site-Specific Recombination
1-3 Serine recombinases introduce double-stranded breaks in DNA and then swap strands to promote recombination
Conservative Site-Specific Recombination
Figure 11-6
Conservative Site-Specific Recombination
1-4 Tyrosine recombinases break and rejoin one pair of DNA strands at a time
Figure 11-7
Conservative Site-Specific Recombination
1-5 Structure of tyrosine recombinases bound to DNA reveal the mechanism of DNA exchange
Cre is a tyrosine recombinaseCre is an phage P1-encoded protein, functioning to circularize the linear phage genome during infectionThe recombination sites of Cre is lox sites. Cre-lox is sufficient for recombinationRead Box11-1 for Cre applicationFigure 11-8
Topic 2 Biological roles of site-specific recombination
The general themes of site-specific recombination
2-1& 2 l integrase works with IHF and Xis to integrate/excise the phage genome into/from the bacterial chromosome
The outcome of l bacteriophage infection of a host bacterium
Establishment of the lysogenic state: requires the integration of phage DNA into host chromosomelytic growth is the growth stage of multiplication of the independent phage DNA that requires the excision of the integrated phage DNA from the host genome.Biological roles of site-specific recombination
Figure 11-2: l genome integration. Recombination always occurs at exactly the same sequence within two recombination sites, one on the phage DNA, and the other on the bacterial DNA.
Bacterial genome
Phage genome
Crossover regions
Int (l-encoded integrase)
Xis (l-encoded excisionase)
IHF (integration host factor encoded by bacteria)
Figure 11-9
l-encoded integrase (Int)
catalyzes recombination between two attachment (att) sites. attP site is on the phage DNA and attB site is on the bacterial genomeIs a tyrosine recombinase, and the mechanism of strand exchange is similar to that catalyzed by Cre recombinase.Requires accessory proteins to assemble the integrase on the att sites. Both IHF and Xis are architectural proteins. IHF binds to DNA to bring together the Int recognition sites. Xis binds to the integrated att sites to stimulate excision and to inhibit integration (see 2-2).2-5 Recombinase converts multimeric circular DNA molecules into monomers
The chromosomes of most bacteria, plasmids and some viral genomes are circular.
During the process of homologous recombination, these circular DNA sometimes form dimers and even multimeric forms, which can be can be converted back into monomer by site specific recombination.
Site-specific recombinases also called resolvases catalyze such a process.
Biological roles of site-specific recombination
Figure 11-14 Circular DNA molecules can form multimers
1.psd2.psdXer recombinase catalyzes the monomerization of bacterial chromosomes and of many bacterial plasmids.
Xer recombinase is a member of the tyrosine recombinase family
Xer is a heterotetramer containing two subunits of XerC and two subunits of XerD. Both XerC and XerD are tyrosine recombinases but recognize different DNA sequence.
The recombination sites in bacterial chromosomes, called dif sites have recognition sites for both XerC and XerD.
Figure 11-15
The dimer only resolves when XerD is activated by the presence of FtsK
Topic 3 Transposition ()
Transposition is a specific form of genetic recombination that moves certain genetic elements from one DNA site to another.These mobile genetic elements are called transposable elements or transposons. Movement occurs through recombination between the DNA sequences at the ends of the transposons and a sequence in the host DNA with little sequence selectivity.FIGURE 11-17 Transposition of a mobile genetic element to a new site in host DNA, which occurs with or without duplication of the element.
Box 11-3 Example of corn cob showing color variegation due to transposition
plant genomes are very rich in functional transposons
Barbara Mc Clintock Maize
The biological relevance of transposons
Transposons are present in the genomes of all life-forms. (1) transposon-related sequences can make up huge fractions of the genome of an organism (50% of human and maize genome). (2) the transposon content in different genomes is highly variable (Fig 11-18).2. The genetic recombination mechanisms of transposition are also used for other functions than the movement of transposons, such as integration of some virus into the host genome and some DNA rearrangement to alter gene expression [V(D)J recombination].
3-(1-6) There are three principle classes of transposable elements
DNA transposons
Viral-like retrotransposons including the retrovirus, which are also called LTR retrotransposons
Poly-A retrotransposons, also called nonviral retrotransposons.
Transposition
FIGURE 11-19 Genetic organization of the three classes of transposable elements
3-2 DNA transposons carry a transposase gene, flanked by recombination sites
Recombination sites are at the two ends of the transposon and are inverted repeated sequences varying in length from 25 to a few hundred bp.
The recombinase responsible for transposition are usually called transposases or integrases.
Sometimes they carry a few additional genes. Example, many bacterial DNA transposons carry antibiotic resistance gene.
Transposition
3-3 Transposons exist as both autonomous and nonautonomous elements
Autonomous transposons: carry a pair of terminal inverted repeats and a transposase gene; function independently
Nonautonomous transposons: carry the terminal inverted repeats but not the functional transposase; need the transposase encoded by autonomous transposons to enable transposition
Transposition
3-4 Viral-like retrotransposons and retroviruses carry terminal repeat sequences and two genes important for recombination
Inverted terminal repeat sequences for recombinase binding are embedded within long terminal repeats (LTRs), being organized on the two ends of the elements as direct repeats.
reverse transcriptase (RT), using an RNA template to synthesize DNA.
integrase (the transposase)
Transposition
3-5 Poly-A retrotransposons look like genes
Transposition
Do not have the terminal inverted repeats.
On end is called 5 UTR (untranslated region), the other end is 3 UTR followed by a stretch of A-T base pairs called the poly-A sequence. Flanked by short target site duplication.
Carry two genes. ORF1 encodes an RNA-binding proteins. ORF2 encodes a protein with both reverse transcriptase (RT) and endonuclease activity. Truncated elements lacking complete 5 UTR??
3-(7-9) DNA transposition by a cut-and-paste mechanism (non-replicative mechanism)
Multimers of transposase binds to the terminal inverted repeats of the transposons, and bring two ends together to form a stable protein-DNA complex called the synaptic complex/transpososome.
This complex ensures the DNA cleavage and joining reaction, which is called strand transfer and is similar to the recombinase
Transposition
FIGURE 11-20 The cut-and-paste mechanism of transposition
One-step transesterification
3-10 DNA transposition by a replicative mechanism/replicative transposition
The mechanism is similar to the cut-and-paste transposition.
The assembly of the transposase protein on the two ends of the transposon DNA to generate the transpososome.The transposase first cleaves one DNA strand at each end of the transposon, resulting in two 3OH ends. BUT NO cleavage occurs at the second strand.
Transposition
Replicative transposition frequently causes chromosomal inversions and deletions that can be highly detrimental () to the host cell.
FIGURE 11-22 Replicative transposition
1st step of replicative transposition : assembly of the transposase protein on the 2 ends of the transposon DNA to generate a transpososome essential to coordinate the DNA cleavage and joining reactions on the 2 ends of the transposons DNA
2nd step : DNA cleavage at the ends of the transposons DNA catalyzed by the trasnposase within the transpososome
The 3OH ends of the trasnposon DNA are then joined to the target DNA site by the DNA strand transfer reaction. The mechanism is the same as cut-and-paste transposition
However, the intermediate generated by DNA strand transfer is in this case a doubly branched DNA molecule
In this intermediate, the 3ends of the transposon are covalently joined to the new target sitre, while the 5ends of the transposon sequence remain joined to the old flanking DNA
Replicative transposition frequently causes chromosomal inversions and deletions that can be highly detrimental to the host cell
This propensity to cause rearrangements may put replicative transposons at a selective disadvantage
Perhaps this is why so many elements have developed ways to excise completely from their original DNA location prior to joining to a new DNA sire
By excision, transposons avoid generating these major disruptions to the host genome
3-11 Viral-like Retrotransposons & Retroviruses move using an RNA intermediate
The mechanism is similar to the DNA transposons (Cut-and-Paste). The major difference is the involvement of an RNA intermediate.
Transcription of the retrotransposon (or retroviral) DNA sequence into RNA by cellular RNA polymerase, which is initiated at a promoter sequence within one of the LTRs.Transposition
Figure 11-23 Mechanism of retroviral integration and transposition of viral-like retrotransposons.
3-12 DNA transposases and retroviral integrases are members of a protein superfamily
Transposition
FIGURE 11-24 Similarity of catalytic domains of transposases and integrases. (a) structure of the conserved core domains of three transposases and integrase
MuA
Tn5
RSV integrase
FIGURE 11-24 Similarity of catalytic domains of transposases and integrases. (b) Scematic of the domain organization of the above three proteins
3-13 Poly-A Retrotransposition move by a reverse splicing mechanism
Transposition
Using an RNA intermediate but a different mechanism from that of the viral-like retrotransposons
The mechanism used is called target site primed reverse transcription.
Transcription of the integrated DNA
The newly synthesized RNA is exported to cytoplasm to produce ORF1 and ORF2 proteins, which remain to bind the RNA
Key points
Conservative Site-Specific Recombination (concept, three types, mechanisms-serine and tyrosine recombinases)
Biological Roles of Site-Specific Recombination (l phage integration/excision, multimeric genome resolution)
Transposition ( concepts, learning from B. McClintock, DNA tranposons. Viral-like retrotransposons/retroviruses, poly-A retrotransposons)